Abstract

MicroRNAs (miRNAs) are small, single stranded non-coding RNAs, 19–24 nucleotides long, involved in crucial biological processes, including differentiation, apoptosis and proliferation. Recent evidence indicates that miRNAs may play an important role in tumorigenesis; changes in miRNAs expression level were identified in many types of human hematological and solid malignancies. To date, several papers have reported the occurrence of genomic deletions flanking the breakpoint on der(9)t(9;22) in 10%–18% of patients with chronic myeloid leukemia (CML). The most probable consequence of der(9) deletions is the loss of tumour suppressor genes, conferring a proliferative advantage to the Philadelphia-positive clone. On the other hand, two miRNAs, namely miR219-2 and miR-199b, are found to map centromeric to the ABL gene within the chromosomal region at 9q34 that is frequently lost in CML patients with der(9) deletions.

In this study, we investigated the loss of miR-219-2 and miR-199b by fluorescence in situ hybridization (FISH) analysis with specific bacterial artificial chromosome (BAC) probes in 68 CML cases bearing der(9) deletions. We further evaluated miR-219-2 and miR- 199b expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) experiments in cases showing deletions of at least one of these miRNAs. Depending on RNA sample availability, miRNAs expression level was evaluated in 7 and in 5 CML cases with miR-219-2 and miR-199b deletions, respectively. Statistical analysis of the relative expression results was performed by the Relative Expression Software Tool (REST). To explore the predicted miR-199b target genes, the miRGen targets database (http:// www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi) was queried; this interface provides integrated data of four widely used target prediction programs (miRanda, PicTar, TargetScan, DIANA-microT).

FISH experiments revealed the loss of miR-219-2 and miR-199b in 17 (25%) and 10 (15%) out of 68 patients. The miR-199b expression study showed a downregulation in the analyzed group of 5 CML cases with miR-199b deletion as compared to a pool of 10 patients without deletions. The expression level of the miR-199b was 0.279 and the difference between the two groups was statistically significant (p= 0.028). On the contrary, the miR- 219-2 analysis did not reveal a detectable expression level in the examined patients. There were 26 predicted miR-199b target genes, involved in several biological processes such as signal transduction (Protein phosphatase inhibitor 2, PPP1R2), regulation of transcription (Hepatic leukaemia factor, HLF), chromosome organization and biogenesis (Zinc finger protein 238, ZNF238), cell proliferation (Mitogen-activated protein kinase 11, MAP3K11) and DNA repair (UV excision repair protein RAD23 homologue B, RAD23B). Among the CML patients evaluable for the response to the treatment, all cases with the miR-199b deletion were resistant to IFN-a and imatinib therapy. In conclusion, our data demonstrate a crucial role for miR-199b in CML cases bearing der(9) deletions. This miR-199b downregulation could influence the expression level of different target genes modifying important cellular pathways. Further analysis of miR-199b target genes will be needed to shed light on the link between miRNAs and CML.

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